Structural and functional characterization of the intrinsically disordered Unique domain of c-Src
Year: 2014
Type: dissertation
Abstract: Objectives: \n \nObjective 1 – Effect of mutations perturbing the ULBR of the Unique domain of c-Src. \n \nThe first objective of this thesis was the in vitro characterization of the effect of mutations in the ULBR on the previously determined main interactions of the Unique domain: binding to lipids and to the SH3 domain. \n \nObjective 2 - Phosphorylation of c-Src Unique domain in Xenopus laevis oocytes and mammalian cell extracts. \n \nPhosphorylation of the Unique domain of c-Src is one of the modulation parameters affecting lipid binding and had previously been reported to affect c-Src activity. \nThe second objective was to study the phosphorylation of the isolated Unique domain in Xenopus laevis oocytes, that enabled the study of phosphorylation in a living cell and in cell extracts, that allowed further manipulation of the phosphatase/kinase network. \n \nObjective 3 - Functional studies of the Unique domain in the context of the full-length protein. \n \nThe last objective was the evaluation of the functional significance of the modifications investigated in the isolated Unique domain as objectives 1 and 2 in a human colorectal cell line and in the context of the full length protein. \n \nThe present thesis addressed fundamental aspects of the biological role of the intrinsically disordered Unique domain of c-Src that until now was poorly understood. We have first characterized through a structural biology approach the effects of sitedirected ULBR mutations on the previously described interactions with lipids and with the folded SH3 domain. Then, we have investigated the phosphorylation events taking place on the Unique domain by state-of-the-art in-cell NMR techniques. Finally, we have shown the functional role of USrc in the context of normal and cancer cell lines. All together, our results have demonstrated that the Unique domain of human c-Src actively participates in the regulation mechanism in which the tyrosine kinase c-Src is involved. This study represents a new essential milestone in the understanding of how c-Src can control a wide variety of signaling events inside cells. Furthermore, it potentially opens the way to the possibility of new applications for cancer therapy through the development of novel selective drugs targeting functional active residues locating in the Unique domain. \n \nThis thesis represents a “proof-of-concept” of how from the atomistic comprehension of a subject of interest (structural approach) it can be possible to understand and successively demonstrate the biological significance of the observed phenomena (functional approach). \n \nConclusions: \n \n-\tMutations in the Unique Lipid Binding Region affect the lipid binding capability of USrc but not the inter-domain interaction with the folded SH3. \n-\tDisruption of the conserved FGGF motif causes important local effects that affect the structure of the ULBR. \n-\tThe SH3 domain of human c-Src loses its ability to interact with lipids in presence of a polyproline peptide. \n-\tReal-time NMR spectroscopy allowed the study of complex phosphorylation/dephosphorylation processes in USrc mediated by kinases and phosphatases in Xenopus laevis derived cells or cell extracts. \n-\tThree different phosphorylation sites present in the Unique domain that were \nunequivocally assigned to Ser 17, Ser 69 and Ser 75 have been identified in Xenopus laevis, COS-7, MEFs and HeLa cell systems. \n-\tIt was the first time that phosphorylation of Ser 69 of c-Src was observed in those model systems. \n-\tThe biological relevance of the ULBR has been firstly demonstrated in Xenopus laevis oocytes in the context of the full-length c-Src. \n-\tThe functional roles of the described phosphorylation events together with ULBR mutations have been shown in HEK293T cells and in the human colorectal SW620 cancer cell lines in the context of the full-length protein. \n-\tA “positional regulation model” has been proposed as a new c-Src selectivity regulation mechanism.
Author: Mariano Maffei
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W646618506